An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance

Abstract

Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

Light‐induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures

“…All S. cerevisiae strains were derivatives of the S288C strains ESM356-1, YJT23, and YJT24. [45,46] Strains are listed together with their rele-vant genotypes in Supplementary Table S1. CRISPR/Cas9 based integration of PCR products was done as described.…”

A modular toolbox for the optogenetic deactivation of transcription

“…The modularity of these two-hybrid systems allows for easy modifications by exchanging individual components. Existing tools include both blue-and red light-responsive dimerizers that have been combined with different DBDs and transactivator domains, depending on the application context 2,3,[9][10][11][12] . In particular, the repurposing of CRISPR nucleases, such as Cas9, as DNA-binding modules has increased the versatility of these systems by enabling the simple and efficient targeting of endogenous loci 2,3,5 .…”

C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways

“…Controlled exposure was achieved with an established blue light switch, a variant of the photoreceptor domain light–oxygen–voltage 2 (LOV2) of Avena sativa phototropin 1 ( 25 ). This domain and relatives thereof have been used previously in the context of targeted protein degradation in diverse cell lines and organisms ( 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ). Light-induced unfolding of an α-helix at the C terminus of the LOV2 domain can be used to expose an otherwise inaccessible degron sequence ( 30 , 34 ).…”

“…1 A ). Similar STFPRs have been used previously to characterize protein degradation efficiency ( 25 , 29 , 35 ). The photoreceptor domain of psd3 (iLID variant of LOV2 with deletion of alanine 416 ( 25 ) was placed at the C terminus of mScarlet I followed by 16 randomly chosen amino acids, which were supposed not to adopt a stable secondary fold.…”